This invention relates to methods and kits for detecting or assaying an analyte present in a water immiscible solvent.
The terms xe2x80x9cwater immiscible solventxe2x80x9d and xe2x80x9caqueous solutionxe2x80x9d are intended to be construed broadly, to include liquids which are partially miscible on agitation but which on settling separate into two layers to form a liquid/liquid interface.
Ligand binding assays have been applied extensively to the detection and quantisation of compounds of biological importance present in fluids such as serum. Such assays rely on the interaction between the compound to be analysed (the analyte) and a specific binding partner such as an antibody. The extent of the binding reaction is generally monitored by the use of a marker, such as a radioactive label, which can be detected at very low concentrations. These assay techniques have been used widely for the measurement of water soluble substances such as proteins and also in the quantisation of compounds such as steroids or thyroid hormones, which are only sparingly soluble in water but which are normally associated with specific or non-specific binding proteins. Assay of these latter compounds frequently involves extraction of the substance of interest into a water immiscible solvent, removal of the solvent by evaporation and then xe2x80x9csolubilisationxe2x80x9d of the compound in an aqueous medium to provide essentially a single phase system in which reaction with a binding partner such as an antibody can occur. Such techniques are labor intensive and can only be performed in specialised laboratories.
The progressive devolution of ligand binding assays to non-specialist laboratories and their increasing use in analytical applications outside clinical diagnostics require simplification of the basic analytical procedures to make them accessible to non-specialist staff in what might be regarded as a xe2x80x9cfieldxe2x80x9d situation.
It has generally been considered that the assay system should essentially comprise a single liquid phase, and as will be evident from the prior art, complex and expensive procedures have been implemented to render a sample of an analyte soluble in an organic solvent suitable for analysis by ligand binding assays.
However, we have discovered, surprisingly, that an effective ligand binding assay may be set up in a two-phase liquid system in which the binding partner is in an aqueous phase and the analyte is in a substantially water immiscible solvent phase and in which binding takes place at or near the interface between the liquids.
The concept of a physiologically derived molecule such as an antibody retaining its activity under conditions where it is exposed to an organic solvent is both unexpected and unpredictable. Russell et al (Biochem Biophys Res Comm 1989; 158: 80) have shown that immobilised anti-hapten antibodies will bind hapten in the presence of water miscible solvents such as dioxane and acetonitrile. However, their experiments showed that the binding affinity of the antibodies was significantly reduced by the presence of the solvent and also that the binding was progressively reduced as the hydrophobicity of the solvent used was increased.
Subsequently, Weetall (J Immunol Meth 1991; 136:139) found that antibodies linked to paramagnetic particles were capable of retaining binding activity in water immiscible solvents such ar hexane.
Francis and Craston (Analyst 1994; 119:1801) developed an immunoassay for parathion in hexane in which the antibody was encapsulated in reverse micelles. In this way, the antibody was xe2x80x9cprotectedxe2x80x9d by inclusion in the micelle.
Recently, Matsuura et al (J Biochem 1993; 114:273) have described the binding of a shell fish toxin to antibodies in the presence of methanol, though they did not develop a quantitive analytical procedure.
U.S. Pat. No. 4,238,472 describes a method in which samples are extracted with a water immiscible solvent which is subsequently removed by evaporation. The dried residues are taken up in a detergent which will form an emulsion with the assay reagents, essentially producing a single phase liquid system.
Published PCT Application No. WO 92/12427 describes an assay procedure where the sample is extracted into hexane and, after further purification, the hexane is removed by evaporation and the sample re-dissolved into a water soluble solvent (methanol). This is then diluted into an aqueous assay buffer, again forming a single phase.
Broadly speaking, the present invention relates to the detection and quantisation of largely water insoluble compounds dissolved in a water immiscible solvent by being brought into contact with a specific binding reagent present in an aqueous medium so that a binding reaction takes place via the water/solvent interface, the extent of the binding reaction, i.e. the degree of association between said analyte and said binding partner being proportional to the concentration of compound in the solvent phase.
Thus, in one aspect of this invention, there is provided a method of determining the presence and/or concentration of an analyte in a substantially water immiscible solvent, which method comprises the steps of:
(i) mixing a sample of the water immiscible solvent with an aqueous solution containing a specific binding partner of said analyte to allow binding between said binding partner and said analyte (if present), and
(ii) monitoring directly or indirectly the degree of association between said analyte and said binding partner thereby to determine the presence and/or concentration of said analyte.
The benefits of this method include the facts that the method provides a two phase liquid system in which binding can occur and that the binding partner is largely preserved or protected by remaining in the aqueous phase, and the natural separate of the aqueous and solvent phases may in many cases simplify separation of the fractions of the bound and unbound analyte.
In a preferred aspect, the binding reagent is an antibody labelled with a substance which allows the extent of binding to be readily assessed.
Preferred embodiments of the present invention allow the development of quantitative or qualitative analytical methods for the detection of hydrophobic compounds present in water immiscible solvents such as hexane, xylene and toluene where the appropriate binding reagent in an aqueous medium is agitated with the solvent sample such that the compound reacts with the binding reagent at or near the water/solvent interface and so becomes effectively xe2x80x9ctrappedxe2x80x9d by the reagent in the aqueous layer. The amount that is trapped in this way is a function of the concentration of analyte in the solvent sample and can be readily determined by standard ligand binding techniques, suitable examples of which are well known to those skilled in the art.
It will be readily appreciated that this approach represents a significant simplification for detecting or assaying an analyte initially present in a solvent, when compared with methods which require prior extraction of the sample or the manufacture of reverse micelles to encapsulate the binding reagent. Such considerations are particularly important when a method is being developed for field use.
In a preferred aspect, the invention involves the use of monoclonal antibodies in solution in an aqueous buffer which are shaken with a sample of a hydrophobic hapten present in a water immiscible solvent. The antibodies, which may be labelled with a marker, are specific for the hapten which will not normally partition into the aqueous phase. After shaking for a predetermined period of time, the mixture is allowed to stand briefly to allow the aqueous and water immiscible solvent layers to separate. The aqueous layer is transferred to a reaction tube which contains an immobilised derivative of the analyte. In a further reaction, labelled antibody which has not reacted with analyte in the sample will bind to the derivative in the tube. The amount of this bound antibody, which varies inversely with the concentration of analyte in the original solvent sample, can be quantified by an appropriate means after it has been washed free of any unreacted antibody. The amount of analyte present in the initial sample can be calculated by reference to reactions of calibrator solutions.
This procedure provides several unique advantages particularly in relation to the establishment of field tests. Firstly, the fact that the binding reagent is free in solution and not immobilised allows for the maximum reaction rate with antigen when the two solutions are agitated together. In practice a reaction time of only one minute may be sufficient to produce significant binding. Secondly, the rate of reaction is concentration dependent so that the measurement of an exact volume of sample is not critical. The avoidance of complex pipetting steps is highly desirable for field testing.
It will also be readily appreciated that simple adaptations of the basic procedure can provide flexibility in the choice of reagents. For example, in another aspect of this invention, it is possible to react an aqueous solution containing unlabelled antibody with the water immiscible analyte solution and then react the aqueous phase with the solid phase analyte as before. After a simple wash, the amount of antibody bound to the solid phase may be detected by means of a further reaction with labelled anti-immunoglobulin antibody. This approach is particularly valuable when only polyclonal antibodies are available, since these may require a complex purification step if they are themselves to be labelled directly.
In another aspect of this invention, a simultaneous reaction for a fixed time period may be performed between labelled antibody in aqueous solution, a water immiscible analyte solution, and the immobilised derivative in a single tube. Once again, the amount of label which becomes associated with the solid phase varies inversely with the concentration of analyte in the solvent sample. This approach adds to the simplicity of the test format, which in turn makes the method suitable for field or laboratory applications.
Though the methods of this invention have been designed for the detection of water insoluble compounds, it is appreciated that the invention can be readily applied even to substances which show some partitioning into aqueous media, on the basis that the degree of partitioning and ultimately of uptake by the binding reagent will still be proportional to the concentration of compound in the solvent. In such a situation, the binding reagent effectively xe2x80x9ctrapsxe2x80x9d the analyte as it transfers from the water immiscible phase to the aqueous phase at the interface. Thus, the method is not restricted in the types of molecule to which it can be applied so long as the analyte substance is present in, or can be extracted into a water immiscible solvent.
There are many important potential applications of this invention. One such application is the identification and quantisation of organic residues present in materials such as soil and water. Pesticides used in agriculture can be difficult to trace in the environment. Moreover, conventional analytical procedures such as mass spectrometry may lack the required sensitivity and in any case are inherently complex. The ability to extract such materials into water immiscible solvents and then quantify them directly by immunoassay provides a simple and general approach to monitoring the presence of potentially toxic compounds present in samples which do not lend themselves readily to analysis by conventional procedures.
The use of solvents for the extraction of analytes from samples such as, for example but without limitation, soil, seeds, foliage and the like, is well-known to those skilled in the art. Such procedures are represented by, for example but without limitation, solvent partitions, the use of solid adsorbents, chromatographic methods and super-critical fluid extraction. The choice of materials and methods for such purposes are well-established and it is well within the scope of one skilled in the art to choose a method appropriate for a particular sample type and analyte.
Thus, in another aspect of this invention, there is provided a method of detecting the presence and/or concentration in a sample of an analyte which is at least partially soluble in a water immiscible solvent, which comprises the steps of:
(i) contacting the sample with said water immiscible solvent to extract said analyte therefrom;
(ii) collecting the water immiscible solvent with any extracted analyte solution;
(iii) mixing said collected water immiscible solvent with an aqueous solution containing a specific binding partner of said analyte to allow binding between said binding partner and said analyte (if present), and
(iv) monitoring directly or indirectly the degree of association between said analyte and said binding partner, thereby to determine the presence and/or concentration of said analyte.
For example, it is difficult to quantify herbicides such as atrazine when they are present at concentrations below 100 ng/ml using conventional chemical approaches. In contrast, a simple immunoassay procedure which can be carried out in less than 10 minutes can yield a sensitivity of detection which is better than 1 ng/ml.
Another important application relates to the ability to identify specifically a manufacturer""s own product in order to combat counterfeiting, as described by Wraith and Britton in EPA 0327163. That application describes the use of compounds which could be added to petroleum based fuels prior to their distribution and subsequently extracted from samples, taken at different points in the distribution process, into an aqueous medium suitable for detection by immunoassay.
In another aspect, this invention provides a means for the direct detection by immunoassay of water immiscible components added to fuel as marker substances. Alternatively, it can be applied to the quantisation of certain additives which are introduced into the fuel to enhance its performance, provided a suitable binding partner for such an additive can be identified. Since certain of these so-called additives are unique to a particular manufacturer, their quantitative assay in this way provides a means of identifying the specific product as well as a means of controlling the quality of the end product to the consumer.
It will be readily appreciated that the ease of use provided by a direct test which does not require complex sample manipulations enables such a test to be performed essentially under field conditions.
The identification of the analyte in question is carried out by means of a specific binding partner such as an antibody. It is appreciated by those skilled in the art that both polyclonal and, more usefully, monoclonal antibodies can be produced to a very wide range of organic compounds. Antibodies can be labelled with a variety of markers such as radioisotopes, enzymes or fluorescent molecules.
In a preferred aspect of this invention, antibodies are labelled with chemiluminescent molecules, preferably acridinium derivatives as described in EP 0082636. Such compounds have several unique advantages for this type of application. Firstly, they can be detected with extremely high sensitivity, thus contributing to the overall sensitivity of the method. Secondly, chemiluminescent molecules are inherently stable until the chemical reaction leading to photon emission is triggered. Thus they can be introduced into a wide range of liquid media without risk of losing chemiluminescence activity.
The analytical procedure can be pursued in a variety of formats as described above. In each case the methods use the fact is that the degree to which the labelled antibody binds to the solid phase derivative is dependent upon the concentration of analyte in the solvent sample. By measuring the degree of binding produced with known analyte concentrations, a calibration curve can be produced to allow the quantisation of unknown samples of the analyte.
Attention has been drawn to one advantage of this type of procedure in that it can be undertaken without the need for complex pipetting steps. Moreover, the light measurement can be carried out with simple, readily available luminometers. Portable instruments are presently available so that in combination with these simplified analytical procedures, it is possible to develop highly sensitive tests for use under field conditions.
Whilst the invention has been described above, it extends to any inventive combination of features set out above or in the following examples.